Cells and biological tissue generally maintain their structure, function, antigen makeup, and chemical constituents within the body. However, once removed from the natural environment of the body, cells and tissue begin to experience immediate changes in these aspects. Thus the preservation and retention of the integrity of cells and biological tissues removed from the body is a continuing challenge in the medical fields, particularly in the fields of blood preservation and organ transplantation.
Circulating whole blood and blood cells are normally quite stable when maintained within the body's environment otherwise disastrous effects would occur due to even small injury. For example, when whole blood is removed from the body, the surrounding environment rapidly changes in parameters such as temperature, pH, and oxygen level. The liquid (plasma) and cellular components in the blood rapidly change in response to changes in temperature and contact with foreign surfaces, such as when drawn into a container. Normally the plasma portion of the blood responds to such change in environmental conditions with the initiation of the coagulation sequence and the subsequent blood clotting. Blood cells, especially those having a physiologic role to most actively respond to environmental changes, lose certain critical physical components and integrity in response to the environmental change. The most sensitive cellular elements in the blood are the short lived blood cells whose function it is to immediately respond to tissue damage and foreign materials such as granulocytes and platelets.
The in vivo state of such cellular components is often represented in the antigen expression of cells as determined by the use of monoclonal antibodies. These antigens are the cluster-designated antigens (CD) present on hematopoietic cells. The appearance or absence of these CD antigens on cells is the basis of cellular identification, characterization and many other clinically useful applications. Changes in the cells are usually initiated by the activation of various cellular enzymes such as phosphatases, protease, oxidases, carboxylase etc. as mediated by (1) a process across the membrane such as receptor binding or surface contact, (2) the exposure to chemicals that are taken up into the cells and cause enzymatic changes such as toxic chemicals, (3) by physical phenomena such as a decrease or increase in temperature. The cellular changes produced in blood cells upon removal from the body are usually represented as the appearance of activated cells, such as are frequently seen with the use of monoclonal antibodies. Thus, the in vivo state of blood is not maintained upon removal from the body.
This tendency of the blood to rapidly change upon withdrawal from the body has proven to be a problem in fields such as cellular analysis and investigational research, and particularly in hematological analysis frequently required for the diagnosis and monitoring of a disease state. For example, it is important to know if the presence of activation associated antigens on blood cells is due to either some aspect of a biological in vivo process or an artifact caused by the in vitro process of obtaining the sample. For accurate research and diagnosis, it is necessary to store and preserve blood and other tissues in a fashion that best represents the in vivo state of that tissue and not the changes caused by removal from the body's environment, such as withdrawal into a container or test tube.
One classical means to prevent changes in whole blood upon removal from the body has been the use of preservatives. The inhibition of the coagulation sequence has been the main objective of conventional anticoagulant preservatives. The classical objective of these anticoagulants for blood analysis was to keep the blood from clotting so that the blood cells could be placed on glass slides for staining and examination. Within the last century these anticoagulants were adapted for use in maintaining certain physical characteristics of the cells such as size and shape. However, while the preservatives maintain cell viability, they nevertheless permit the cells to change in response to the stimulus of the blood drawing process. Thus the use of anticoagulants prevents the plasma from clotting but does not necessarily prevent the changes that occur in the cellular elements. The basis of the most common anticoagulants is the inhibition of coagulation enzymes directly or through the removal of the calcium ions required for many enzymatic processes. Thus blood cells, even in anticoagulated blood, change with time after removal from the body and, when analyzed, may not represent the in vivo state of the blood within the donor. Anticoagulation has most often been accomplished by binding or chelation of calcium ions by a variety of substances such as citrate or ethylenediaminetetraacetic acid (EDTA). Other traditional methods of anticoagulation have involved adding to the withdrawn blood natural enzymatic inhibitors of the coagulation sequence such as heparin or sodium fluoride or hirudin. Such anticoagulants or preservatives, while commonly used for maintaining blood in a liquid state and for the isolation of viable cells for culture, are not designed for maintaining cellular antigens in a representative in vivo state. The longer the cells remain in the anticoagulant the more the cells change due to the effects of the anticoagulant and the effects of storage.
Another approach to blood and tissue preservation has been the use of “fixative” solutions, such as formaldehyde and glutaraldehyde, which react with and cross link the proteins causing them to denature and become insoluble. A variation of this process occurs with the use of dehydrating alcohols, picric acid, mercuric compounds, tannic acid and many other compounds. These historic processes, which are variations of the process for making leather, irreparably alter the structure of proteins. Antigen structures (epitopes) dependent upon defined protein structure are usually destroyed and so is the corresponding ability to react with epitope specific monoclonal antibodies. Even at low concentrations, these fixatives cause cellular changes ranging from the loss of membrane integrity and the lysis of red blood cells to the up-regulation of surface activation antigens.
In a known method of blood and tissue preservation, whole blood is treated with a composition containing a mixture of ammonium-chloride to lyse the red blood cells, enzyme inhibitors and paraformaldehyde. The preserved white blood cells are washed and separated out, thereby producing Beckman Coulter's Immuno-Trol™ cell control product, which maintains the antigenicity of the white blood cells.
With the advent of automated cellular analysis equipment, such as Flow Cytometry, and the invention of monoclonal antibodies, the identification of specific cellular markers on cells and the analysis of cellular attributes and their impact on disease has become a vital component of medical diagnosis and resulting treatment of disease. However, although cellular analysis has changed greatly in the last few years there has been little change in the nature of blood preservation.
For example, U.S. patent application Publication No. US2002/0028517 refers to a method for using the electronic impedance cell counting technique for determining platelet activation by comparing the number of platelets in EDTA preserved samples from which is subtracted the number platelets in a second sample activated with a platelet agonist, such as collagen, ACP, epinephrine and ristocetin solutions, in the absence of EDTA and using the difference as a measure of platelet activity.
Similarly, U.S. Pat. No. 5,486,477 refers to a blood diluent that stabilizes blood cells without fixing the permeability of the cell membranes which includes formaldehyde in a concentration range of 0.01% to 0.15%, an organic buffer, water, and sodium chloride and sodium sulfate, with a base to adjust pH. The disclosure states that no additional reagents are necessary to achieve blood cell stabilization or function as an antimicrobial agent, because numerous additives may interfere with leukocyte membrane permeability.
U.S. Pat. No. 5,459,073 refers to a fixative with low toxicity employing a formaldehyde donor, such as diazolidinyl urea, imidazolidinyl urea, dimethylol-5,5-dimethylhydantoin, dimethylol urea and the like rather than formaldehyde itself.
U.S. Pat. Nos. 6,197,540 and 6,197,539 refer to a stabilized blood composition and method that involves treating separating leucocytes from a blood sample, treating the leucocytes with an aged transition metal solution and reintroducing the treated leucocytes back into the blood sample.
Still other efforts to stabilize blood samples and cellular components of blood have varied. For example, U.S. Pat. No. 5,516,695 refers to a multi-reagent system for analysis of whole blood which contains a non-quaternary ammonium salt, an aliphatic aldehyde having 1-4C, a surface active agent, e.g., saponin, a non-phosphate buffer, e.g., an acetate buffer, and water. This reagent system concurrently lyses RBC and fixes WBC while preserving WBC membranes and surface antigens.
There remains a need in the art for methods and compositions for stabilizing blood and tissue upon removal from the body, to prevent and/or reduces cellular activation and response to environmental change without changing the antigenic makeup of the cells. There remains a need in the art to provide improvement over existing anticoagulants and preservatives in the in vivo representation of the blood drawn from a donor.